The sample for the culture may be obtained by suctioning the ulcer active edge or by extracting a fragment of tissue which is then macerated in a phosphate-buffered saline solution (PBS) with antibiotics (1000 UI of crystalline penicillin per cc), before it is put in the culture medium. A tuberculin syringe with a thin needle (26G), containing 0.3 cc of PBS solution with antibiotics is used in the suction technique. Previous asepsis of the ulcer with alcohol at 70%, a needle is introduced into the dermis and through rotating movements a small amount of tissue is macerated by the needle bevel during about a minute, after which it is suctioned into the syringe. The sample is deposited in aseptic conditions into a NNN culture medium and incubated at 26ºC during 4 weeks [46,47]. Every week a drop is extracted from the culture medium and placed between two slides to be observed under a microscope. In case that promastigotes are not found, the cultures are rejected as negative [46]. The strains are identified by species using the monoclonal antibodies developed by Dr. Diane Mc Mahon Pratt [48,49].